Fluorescent polymer superquenching-based bioassays

ABSTRACT

A chemical composition including a fluorescent polymer and a receptor that is specific for both a target biological agent and a chemical moiety including (a) a recognition element, (b) a tethering element, and (c) a property-altering element is disclosed. Both the fluorescent polymer and the receptor are co-located on a support. When the chemical moiety is bound to the receptor, the property-altering element is sufficiently close to the fluorescent polymer to alter the fluorescence emitted by the polymer. When an analyte sample is introduced, the target biological agent, if present, binds to the receptor, thereby displacing the chemical moiety from the receptor, resulting in an increase of detected fluorescence. Assays for detecting the presence of a target biological agent are also disclosed.

This application claims priority from U.S. Provisional Application Ser.No. 60/276,090 filed Mar. 16, 2001 and U.S. Provisional Application Ser.No. 60/314,101 filed Aug. 23, 2001. The entirety of those provisionalapplications is incorporated herein by reference.

BACKGROUND OF THE INVENTION Field of the Invention

The present invention relates to a fluorescent biosensor that functionsby a novel Quencher-Tether-Ligand (QTL) mechanism. In particular, thepresent invention relates to improving the polymer-QTL approach byco-locating the fluorescent polymer (or polymer ensemble, includingself-assembled polymers) and a receptor for the QTL bioconjugate andtarget analyte on the same solid support.

DISCUSSION OF THE BACKGROUND

The polymer-QTL (Quencher-Tether-Ligand) approach is a single-step,instantaneous, homogeneous assay where the amplification step isintrinsic to the fluorescent polymer. The polymer-QTL approach providesa system for effective sensing of biological agents by observingfluorescence changes. The key scientific basis is the amplification ofquenching of fluorescence that can be obtained with certain chargedconjugated polymers and small molecule quenchers. In addition, theprocess is uniquely simple because there are no reagents.

In the “biosensor” mode, the QTL approach functions by having afluorescent polymer quenched by a specially constructed“quencher-tether-ligand” (QTL) unit as shown in the diagram set forth inFIG. 1. Addition of an analyte containing a biological receptor specificto the ligand is expected to remove the QTL conjugate from the polymerwhich results in a “turning on” of the polymer fluorescence. Afluorescent polyelectrolyte-based superquenching assay has been shown tooffer several advantages over conventional small molecule basedfluorescence assays. For example, conjugated polyelectrolytes,dye-pendant polyelectrolytes, etc. can “harvest” light effectively bothby absorption and by superquenching (1-5). The enhanced absorbing powerof the polymers is indicated by the observation that even sub nanomolarsolutions of some of these materials are visibly colored. Thefluorescence of these polymers can be detected at even lowerconcentrations. Superquenching occurs in the presence of small moleculescapable of serving as electron transfer or energy transfer quenchers tothe polymer or one of its repeat units.

The “Stern-Volmer” quenching constants (K_(SV)) for these polymers havebeen shown to be as high as 10⁸-10⁹ M⁻¹, and it is anticipated thatvalues as high as 10¹¹ M⁻¹ may be attainable (6). Such high values forK_(SV) toward quenchers oppositely charged to the polyelectrolyte areinitiated by strong nonspecific binding between the quencher and thepolyelectrolyte. Subsequent amplified quenching occurs due to acombination of excitonic delocalization and energy migration to the“trapsite” where the quencher is in close proximity with the polymer.

It has also been shown that enhanced superquenching may be obtained whenthe polymers are adsorbed onto charged supports including surfaces,polymer microspheres, and inorganic nanoparticles (7,8). Superquenchinghas also been observed in the same supported formats for monomers orsmall oligomers self-assembled into “virtual” polymers. Polymer (and“virtual” polymer) superquenching has been adapted to biosensing byconstructing QTL conjugates containing a potential superquenchingcomponent (Q) tethered (T) to a bioreceptor (L) or ligand for a specificbiomolecule (1).

A fluorescence based assay is realized when the QTL conjugate is used toquench the polymer either in solution or in supported formats atsolution-solid or solution-particle interfaces (1,7,8). For example,fluorescent polyelectrolytes, including conjugated and J-aggregatepolymers, can be used for sensitive biodetection and bioassays insolution formats. The basis of this detection is the combination of the“superquenching” sensitivity of these molecules to quenchers of oppositeor neutral charges with the synthesis of a quencher-recognitionconjugate (e.g., a QTL molecule). In the original formulation, the QTLconjugate quenches the polymer ensemble by nonspecific binding. Additionof a target bioagent capable of binding with the L component of the QTLconjugate results in a removal of the QTL conjugate from the polymer anda turning on of the polymer fluorescence.

A fluorescence turn off (or modulation) assay has also been developedbased on polymer superquenching (5). In this case, the target moleculeis a bioagent L, or L′, corresponding to the L component of the QTLconjugate, and the receptor is a biomolecule that strongly associateswith L, L′ or the QTL conjugate. One example is a direct competitionassay in which L (or L′) in unknown amount is allowed to compete withthe QTL conjugate for the binding sites of a measured amount of thereceptor. The polymer fluorescence is quenched by non-bound QTL to anextent depending on the amount of L (or L′) present. In another example,the QTL conjugate is preassociated with the receptor; when all of theQTL conjugates are associated with the receptor sites, no quenching isobserved. Addition of L (or L′) to the sample results in the release ofthe QTL conjugate with concomitant quenching of the polymerfluorescence.

All of the above assay formats depend on nonspecific quenching of thepolymer fluorescence by association of the QTL conjugate with thepolymer. A complication with these assays is the competing nonspecificinteractions of other components of the assay sample with either thepolymer, the QTL conjugate, or both, which result in a modulation of thequenching. In the present invention, modifications of the polymersuperquenching allow the construction of improved assays which overcomethese effects and provide for a more versatile and robust sensor.

SUMMARY OF THE INVENTION

It is an object of the invention to provide a novel chemical moietyformed of a quencher (Q), a tether (T), and a ligand (L) specific for aparticular bioagent.

It is another object of the invention to provide an assay to detect atarget agent in a sample using the novel QTL molecule of the presentinvention and a fluorescent polymer.

It is yet another object of the invention to rapidly and accuratelydetect target biological agents in a sample.

It is a feature of the invention that the fluorescent polymer and thereceptor for the target biological agent are co-located on a support.

It is another feature of the invention that the co-located fluorescentpolymer and the receptor are tethered to the support.

It is yet another feature of the invention that the co-locatedfluorescent polymer and receptor are covalently linked to the support.

It is also a feature of the present invention to covalently link thereceptor to the fluorescent polymer.

It is a further feature that the change in fluorescence is indicative ofthe presence of the target biological agent.

It is another feature of the invention that the quench event is a resultof a specific interaction between the receptor and the QTL conjugate.

It is yet another feature of the present invention that the assembledmonomers behave like polymers.

It is an advantage of the invention that the assays of the presentinvention can be carried out in operationally different formats.

A further advantage of the invention is the versatility provided by theability to control the co-located assembly of a specific polymerensemble-receptor either spatially as on a rigid support or on differentparticles.

It is another advantage of the present invention that assays accordingto the present invention are both homogeneous and near instantaneous.

It is yet another advantage of the invention that the ability to controlthe co-located polymer assembly either spatially (e.g., on a rigidsupport) or on different particles offers great versatility.

It is a further advantage that superquenching occurs due to specificligand-receptor interactions.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a general illustration of the QTL approach.

FIG. 2 illustrates various fluorescent compounds, quenchers, and QTLconjugates used in the present invention.

FIG. 3 illustrates various fluorescent compounds, quenchers, and QTLconjugates used in the present invention.

FIG. 4 illustrates structures of dyes used with polysaccharides ininclusion complexes.

FIG. 5 is a general illustration of a displacement competition assay.

FIG. 6 is a general illustration of a direct competition assay.

FIG. 7 is an illustration of the competitive fluorescence “turn-on”assay with the polymer-biomolecule combination.

FIG. 8 illustrates the co-location of a polymer and a receptor by acovalent/adsorption sequence.

FIG. 9 illustrates the covalent tethering of both the polymer and thereceptor binding site.

FIG. 10 illustrates a receptor covalently linked to a polymer.

FIG. 11 is an illustration of a sandwich QTL assay.

FIG. 12 illustrates various compounds used in the examples of thepresent invention.

FIG. 13 is a graphical illustration of the quenching of fluorescence asa function of the loading level.

FIG. 14 is a graphical illustration of a competition assay for goatanti-rabbit IgG antibody.

FIG. 15 is a graphical illustration of an IgG assay with polymer 25linked covalently to a receptor.

FIG. 16 illustrates the synthesis of cyanine dye 26 covalently appendedto a silica microsphere surface.

FIG. 17 is an illustration of the structure of QSY-21 SuccinimidylEster.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

A key scientific basis for the polymer QTL approach is the amplificationof quenching of fluorescence (superquenching) that may be obtained withcertain polymers (including, but not limited to, charged polymers,conjugated polymers and dye-pendant polyelectrolytes in which thechromophores are collected by non-covalent interactions (e.g.,J-aggregation)) and small molecule quenchers. The fluorescent polymersprovide amplification over conventional molecular fluorophores both byvirtue of their light-harvesting properties (collective excitation) andtheir sensitivity to superquenching (i.e., one quencher may extinguishluminescence from an entire polymer chain or a collection of polymers,oligomers, or monomers). In some cases, enhanced quenching may beobserved when mixtures of polymers are used or when the polymers areadsorbed or otherwise assembled onto surfaces. The same enhancement ofquenching can be observed when monomers or oligomers of some of thechromophore repeat units are assembled either by covalent attachment oradsorption onto a support. The support may be, but is not limited to,any of the following: polymer or silica microspheres, organic orinorganic nanoparticles, magnetic beads or particles, semiconductornanocrystals, tagged or luminescent particles, membranes and planar orcorrugated solid surfaces.

Fluorescent polymer superquenching has been adapted to biosensingapplications through the use of “QTL” bioconjugates (1, 4-6, 8). The QTLapproach to biosensing takes advantage of the superquenching offluorescent polyelectrolytes by electron transfer and energy transferquenchers. In its simplest approaches, the fluorescent polymer, P, formsan association complex with a QTL bioconjugate, usually one with theopposite charge of P. QTL bioconjugates include a small moleculeelectron transfer or energy transfer quencher (Q), linked through acovalent tether to a ligand, L, that is specific for a particularbioagent or receptor. The binding of the QTL bioconjugate by thebioagent either removes the QTL bioconjugate from the fluorescentpolymer, or modifies its quenching efficiency, thus allowing sensing ofthe bioagent in a readily detectable way.

Suitable examples of ligands that can be used in “QTL” methods includechemical ligands, hormones, antibodies, antibody fragments,oligonucleotides, antigens, polypeptides, glycolipids, proteins, proteinfragments, enzymes, peptide nucleic acids (PNAs), and polysaccharides.Examples of suitable tethers include, without limitation, single bonds,single divalent atoms, divalent chemical moieties of up to approximately100 carbon atoms in length, multivalent chemical moieties, polyethylene,polyethylene oxides, polyamides, non-polymeric organic structures of atleast about 7-20 carbon atoms, and related materials. Suitable quenchersinclude methyl viologen, quinones, metal complexes, fluorescent andnonfluorescent dyes, and energy accepting, electron accepting, andelectron donating moieties. These examples of the ligand, tetheringelements, and quenchers are not to be construed as limiting, as othersuitable examples would be easily determined by one of skill in the art.

Polymer-Polymer Ensembles and their Application to Biosensing

The fluorescent polyelectrolytes typified by compounds 1 and 2 in FIG. 2show, in addition to their adsorption properties, a very strong tendencyto associate with oppositely charged macromolecules, including otherpolyelectrolytes and each other. Cationic conjugated polymer 8, togetherwith compounds 1 and 2, form a series of three fluorescentpolyelectrolytes with absorption maximum wavelengths that span the rangefrom the near ultraviolet to the visible-infrared, especially by varyingthe cyanine substituent in compound 2.

The association of two oppositely charged fluorescent polyelectrolytescan lead to several interesting and potentially useful effectsconsidering the association of compounds 1 and 2. For example, theassociation of nearly equimolar, in repeat units, amounts of compounds 1and 2 results in an ensemble that is overall close to neutral, yetconsists of discrete regions of negative and positive charges. Sincecompound 2 shows an emission at lower energies than compound 1, it isobserved that energy transfer should occur. Thus, excitation intoregions where the absorption should be primarily by compound 1 resultsin predominant emission by compound 2. Since compound 2 has a very sharpemission, the harvesting of energy within the ensemble providepossibilities to tune both the absorption and emission properties farbeyond that which is available within a single polymer. A most strikingadvantage obtained by using an ensemble such as the combination ofcompounds 1 and 2 is that both anionic and cationic small moleculequenchers can quench the overall near-neutral polymer mixture. As aresult, it is observed that the ensemble is quenchable (independently)by both anionic compound 4 and cationic compound 3. More importantly,the quenching can be observed at very low concentrations of eitherquencher such that the degree of superquenching shows only a slightattenuation compared to quenching of the individual polymers by theoppositely charged small molecule.

These results show that the polymer-polymer approach offers distinctadvantages for biosensing by the polymer-QTL method. The polymerensemble can be quenched by both positive and negatively charged QTLbioconjugates. Therefore, either in quench/unquench formats or in acompetitive assay, the polymer-polymer ensemble provides a means ofobtaining higher selectivity and specificity. Furthermore, the degree ofquenching by either cationic or anionic quenchers can be tuned directlyby varying the stoichiometry of the polymer mixture. For example, whenpolymer 1 and polymer 2 are mixed in a ratio of 100:1, thesuperquenching by cationic QTLs is maintained and no quenching byanionic QTLs is observed. However, efficient energy transfer is stillobserved to polymer 2 even at this low ratio. By going to a 2:1 ratio ofpolymer 1:polymer 2, superquenching by both cationic and anionic QTLs isobserved. Thus, charge tuning of the QTL assay is achieved by alteringthe stoichiometry of the anionic and cationic polymer. Both the netcharge of the supramolecular cluster and the energy transfercharacteristics of the combination may be tuned in this manner.

Multiplexed Detection Using Mixtures Containing Supported Polymer

The interaction of anionic and cationic fluorescent polymers can beeliminated by first anchoring either polymer to a bead or othersupported format. For example, it has been demonstrated that anchoringpolymer 2 to a clay suspension, prior to the addition of polymer 1prevents the association of polymers 1 and 2. In this way, independentsuperquenching of each polymer is achieved in a single solution uponaddition of either cationic or anionic quenchers.

Supported Formats for Monomers, Oligomers and Polyelectrolytes

Fluorescent polyelectrolytes, including conjugated and J-aggregatepolymers, can be used for sensitive biodetection and bioassays insolution formats. The basis of this detection is the combination of the“superquenching” sensitivity of these molecules to quenchers of oppositeor neutral charges with the synthesis of a quencher-recognitionconjugate (QTL). One improvement of the polymer-QTL approach involvesanchoring the fluorescent polymer onto a solid support via adsorption.Several advantages can result from this adsorption.

Fluorescent polyelectrolytes, including, but not limited to, compoundssuch as those shown in FIGS. 2 and 3 may be readily adsorbed fromaqueous or mixed aqueous-organic solutions onto oppositely chargedsurfaces such as slides, plates, oppositely charged polymer beads (suchas, but not limited to, quaternary amine-derivatized polystyrene orsulfonated polystyrene), and natural or synthetic inorganic supportssuch as clays or silica, charged membranes, or other porous materials.Once adsorbed onto these supports, the polymers retain their intensefluorescence as well as their sensitivity to specific quenchers. Thefluorescent polymers incorporated into these formats may be used inadvanced assays as described below.

The incorporation of a fluorescent polymer onto a charged polymer beadcan result in the reversal of the charge specificity in quenching of thepolymer fluorescence as well as in improved performance in assaysinvolving the polymer in either fluorescence quench or fluorescenceunquench modes. In one example, the anionic conjugated polymer 1 iseffectively quenched by low concentrations of the positively chargedelectron acceptor 3 in aqueous solution. However, its fluorescence islargely unaffected in solution by the addition of the negatively chargedelectron acceptor 4. When polymer 1 is treated with a suspension ofquaternary amine (cationic) derivatized polystyrene beads (Source 30 Q),the polymer is removed from solution and is irreversibly adsorbed ontothe beads. In this supported format, the highly fluorescent beads can besuspended in an aqueous solution and treated with the same quenchers. Areversal of the quenching sensitivity is observed; in the supportedformat, the anionic electron acceptor 4 quenches polymer 1, while thefluorescence of polymer 1 is no longer quenched by cationic electronacceptor 3.

The charge reversal of fluorescence quenching can be adapted tobiosensing by the polymer-QTL approach. Thus, QTL conjugate 5, whichcontains an anthraquinone quencher similar to anionic electron acceptor4 and a biotin ligand, is also observed to quench the fluorescence ofpolymer 1. Upon addition of the protein avidin (a specific receptor forbiotin), the quenching produced by conjugate 5 is reversed and virtuallycomplete recovery of the fluorescence of polymer 1 is observed. Thiscontrasts with aqueous solutions where a viologen-based conjugate 6 hasbeen shown to elicit a similar quench-recovery response with polymer 1.For both polymers 1 and 2, when dissolved in aqueous or partiallyaqueous solutions, nonspecific effects are frequently observed upon thepolymer fluorescence by addition of macromolecules, particularlyproteins leading to either partial quenching or enhancement. Theseinteractions may occur with analyte proteins or with proteins notanticipated to interact with the specific QTL conjugate employed in thesensing and may interfere with specific effects due to the interactionof an “analyte” protein with the polymer QTL complex. These nonspecificeffects maybe eliminated or attenuated by employing polymers insupported formats.

A second example involves the use of the QTL conjugate 7, which quenchesthe fluorescence of polymer 1 by energy transfer. While anionic compound7 does not quench the fluorescence of anionic polymer 1 in pure aqueoussolutions, adsorption of polymer 1 on beads results in its quenchingupon the addition of compound 7 and fluorescence recovery upon additionof avidin.

Adsorbing a fluorescent polymer on a charged support may not always leadto charge reversal in the quenching of the polymer. The charge reversal,or lack thereof, can be tuned by the degree of “loading” of the polymeronto sites on the support. In a different example, it is demonstratedthat enhanced quenching can be obtained for a supported polymer as aconsequence of adsorption. Thus, when cationic polymer 2 is adsorbedonto anionic Laponite clay particles, the polymer fluorescence is stillsubject to quenching when small amounts of anionic acceptor 4 are addedto the aqueous suspension. Under these loading conditions, polymer 2 isnot quenched by cationic acceptors such as compound 3. Quantitativeanalysis of the extent of quenching by compound 4 under these conditionsindicates that the clay-supported polymer 2 is quenched more effectively(in this example by more than 30%) than when it is in a pure aqueoussolution. This example illustrates two concepts that lead to improvedbiosensing with the polymer-QTL approach using supported polymers. Thefirst concept is that the supported polymer can be used to “sense”oppositely charged quenchers when supported on the clay particles andyet exhibit improved stability with respect to degradation andprecipitation (observed for aqueous solutions). When the same polymer issupported on the clay at lower loading levels, its fluorescence isquenched by cationic compound 3, thus demonstrating a charge reversalsimilar to that cited above with polymer 1. The second concept fromthese experiments with clay-supported polymer 2 and its quenching bycompound 4 is that increased quenching sensitivity can be obtained dueto polymer-polymer association effects on the clay particles. Thisincreased quenching sensitivity may result from an increase in theJ-aggregate domain (or conjugation length for conjugated polymers).

The combination of enhanced quenching sensitivity and the ability totune the quenching sensitivity in supported formats as described abovegreatly extends the potential of the polymer-QTL approach both inregards to sensitivity and versatility. Additionally, the anchoring offluorescent polyelectrolytes on beads, surfaces, or membranes can expandthe utility of the polymer-QTL approach. Thus, the strong adsorption ofthe polymers onto beads or membranes can provide detection of analytesin a “flow-through” mode using either liquid or vapor streams.Additionally, the tethering of the polymer onto plates in a multi-wellarray format by adsorption demonstrates the use of these formats in highthroughput screening and rapid sampling applications. Furthermore, theelimination of nonspecific effects upon anchoring to a bead surfacegreatly enhances the practical usage of QTL-based assays.

Virtual Polymers based on Covalent Attachment of Supramolecular BuildingBlocks.

Enhanced superquenching provides a new means of obtaining superquenchingfrom much smaller oligomers and even monomers in an adsorbed format.Thus, it is possible to synthesize polymer 2 in a range of repeat unitsizes varying from n=3 to n=1000. It would be anticipated that, to afirst approximation, in solution, the higher molecular weight polymersshould exhibit higher quenching efficiencies due to an “amplificationfactor” that should be directly proportional to the number of repeatunits (6). However, as the number of repeat units increases, thesolubility of the polymer decreases and the complexity of the polymerallows new channels for nonradiative decay to attenuate theeffectiveness of quenchers. Therefore, in the case of polymer 2, thepotential for attaining maximum sensitivity by using very high molecularweight polymers cannot be recognized. The use of smaller oligomers (oreven monomers) in an adsorbed format permits the construction ofeffective higher order polymers by the formation of extended aggregatesthat bridge across adjacent polymer (or monomer or oligomer) molecules.This provides for enhanced levels of superquenching and thus new sensorsof greatly enhanced sensitivity.

Assembly of cyanine dye monomer 15 or oligomers 10 on silica or claynanoparticles results in the formation of “J” aggregates that exhibithigh superquenching sensitivity (i.e., surface activated superquenching)to ionic electron transfer or energy transfer quenchers. This can beattributed to a combination of high charge density (and resultingCoulombic interactions) and excitonic interactions within theself-assembled units. These assemblies also can be used as biosensors inthe QTL fluorescence quench-unquench mode. These virtual polymers can beeasily assembled from a variety of monomer or small building blocks,often bypassing difficult steps of polymer synthesis, purification, andcharacterization. Although studies to date have shown self-assembledvirtual polymers to be relatively stable with little sensitivity intheir fluorescence to added macromolecules, it is clear that the smalladsorbed units may be subject to desorption or rearrangement undercertain conditions, most notably high ionic strength. An approach thatcombines the simplicity of using small building blocks assembled on asurface with a more robust analysis platform involves the covalenttethering of monomers on the surface of a neutral or chargednanoparticle, bead, or other rigid support.

In one example, a relatively simple synthetic scheme similar to thatdeveloped for the cyanine poly-L-lysine 10 was employed in theconstruction of cyanine dye 15 covalently attached to the surface of 0.2μm diameter silica microspheres. The cyanine dye thus linked to themicrosphere surface was found to exist both as small clusters of themonomer and as highly ordered aggregates. Efficient exitonmigration/energy transfer between the dye clusters and aggregates wasobserved when the material was suspended in water containing 2%dimethylsulfoxide. The suspension also showed a 27% reduction inemission intensity in the presence of 27 nM anionic quencher 13,indicating that superquenching of the covalently-linked dye assembliesoccurs. The modes of interaction between cyanine dye monomers on themicrosphere may be controlled by varying the density and structure offunctional groups present on the surface. Thus, the efficiency ofbiosensing can be optimized. Similar schemes may be used to append othercyanine dyes and other building blocks such as conjugated polymeroligomers onto a bead, particle, or other solid surfaces.

Virtual Polymers Appended onto Quantum Dots by Self-Assembly or CovalentTethers: Coupling of Quantum Dots with QTL Bioassays.

The assembly of cyanine dyes (including, but not limited to, thechromophore of structures 10 and 15) or other molecules capable offorming aggregates onto a particle or surface provides a platform forbiosensing based on superquenching. The superquenching can be controlledby the charge of the assembled film or the surface or a combinationthereof. Biosensing may be accomplished either by fluorescence “turn-on”or “turn-off” assays and in direct and competition modes. While theassembly may have relatively strong light-absorbing properties, in anumber of cases, the absorption of J-aggregates is very sharp andlimited to a very narrow portion of the visible spectrum. A significantenhancement of light-harvesting properties may be obtained byconstructing the assembly on top of a layer or particle having strongabsorption (and high oscillator strength) at higher energies. This canbe accomplished in Langmuir-Blodgett Assemblies and complex multilayeredfilms built up by layer-by-layer deposition.

The construction of an assembly of dyes or other molecules on asurface-capped semiconductor nanoparticle “quantum dot” offers aconvenient and effective way of enhancing the biosensing capabilities ofthe virtual polymers described above. Although quantum dots have beeninvestigated for several years, recent advances have made possible thepreparation of quantum dots of high stability, variable size, versatilewavelength tunability for both absorption and emission properties, andcontrolled surface properties and functionality. Thus, it is possible touse an appropriately constructed and derivatized quantum dot as asupport on which to construct a virtual polymer. The quantum dot“platform” is selected to have good energy donor properties towards aspecific cyanine dye, cyanine dye aggregate, conjugated polymeroligomer, or other building block that can be used in a QTL bioassay.The combination affords an attractive, versatile, yet relatively simpleway of enhancing the sensitivity and extending the wavelength range ofthe QTL biosensor. Both direct adsorption onto the quantum dot orcovalent attachment or anchoring of dots and polymers on a microspheresurface may be used to construct the quantum dot-sensing ensemble.Examples of quantum dots include (but are not limited to) CdS, CdSe andZnS.

QTL Bioassays Based on Assemblies and Inclusion Complexes of DyeMonomers, Oligomers, and Conjugated Polymer Oligomers in Natural andFunctionalized Polysaccharides.

A wide range of investigations have shown that the starch-derivedpolymers amylose and carboxymethylamylose (CMA), which consist oflinear, unmodified or derivatized 1,4 glucose polymers, can formcomplexes with hydrophobic or amphiphilic molecules that can exist asmoderately linear conformations. The complexed “guest” amphiphilesexhibit restricted mobility and, in some cases, a degree of protectionfrom other reagents present in the same solution with the amylose (orCMA) and its guest. The entrapment is attributed to formation of ahelical sheath of the glycoside which surrounds a guest within thecavity. Helices with different radii can be formed to entrap guests ofdifferent sizes. Unmodified amylose is overall neutral while CMA (whichis reasonably easily synthesized with variable loading of thecarboxymethyl groups) is anionic. Analogous derivatization processes arepossible to prepare amylose derivatives with other functionalitiesand/or charge. Several amphiphilic or hydrophilic moleculesincorporating dyes or aromatic chromophores and exhibiting lowsolubility in water or aqueous-organic mixtures can be solubilized inamylose or CMA solutions with the guest chromophores entrapped withinamylose (or CMA). Among examples of the latter are photo- andthermochromic dyes, highly luminescent stilbene amphiphiles, and otherphotoreactive compounds.

Amylose, CMA, and other polysaccharides can form complexes with stronglyabsorbing amphiphilic molecules including appropriately derivatizedsquaraine dyes, bissquaraines, and some conjugated polymer oligomerssuch as poly(phenyl)ethynyl oligomers. Structures of some of thesecompounds selected are shown in FIG. 4. In each case, the compounds areeither actually or potentially highly fluorescent in homogeneoussolution. Additionally, they are either insoluble in water or veryslightly soluble. Structurally they are able to form complexes witheither amylose, CMA, or other modified amylose polymers. Whenincorporated with a charged amylose polymer, they become soluble inwater, strongly fluorescent, and somewhat protected from association(such as face-to-face interactions which quench fluorescence) andadventitious quenching by nonspecific interactions with other solutes.The ability of the amylose and CMA hosts to collect multiple guestsallows the gathering of several molecules of the host chromophores shownin FIG. 4. The high oscillator strength of the chromophores allowsexcitonic interactions to occur even when the chromophores are not indirect contact. These excitonic interactions provide a way of forminganother “virtual polymer” similar to those described above. This virtualpolymer may be subject to quenching by electron transfer or energytransfer quenchers that are brought into close proximity with theamylose or CMA helix containing the guest dyes or oligomers. Thisassociation may be obtained through Coulombic interactions between thequencher and complex or by other interactions that lead to strongassociation. Targeted superquenching by these quenchers can thus beobtained for included molecules such as those shown in FIG. 4, even whenthe individual molecules are not subject to superquenching. As describedabove, it is straightforward to extend superquenching to the use of QTLbioconjugates and to apply these bioconjugates in extensions of the QTLfluorescence quench-unquench and competitive assay formats.

The present invention is a further extension of the use ofsuperquenching in biosensing. By co-locating a bioreceptor and afluorescent polymer (or “assembled polymer”) on a surface or colloidalparticle, the interaction between the two components (quencher (Q) ofthe QTL and the polymer ensemble) is rendered a specific interaction bythe ligand-receptor binding. Thus, the assay is not dependent uponnonspecific charge-based interactions between the quencher and thepolymer ensemble. An additional advantage of the present invention isthe versatility afforded by the ability to control the co-locatedassembly of a specific polymer ensemble-receptor either spatially (forexample, on a rigid support) or on different particles. This greatlyexpands the ability of the QTL approach to be used for multiplexingseveral target agents.

All of the assay formats of this invention rely on a co-location of afluorescent polymer (or fluorescent “self-assembled” polymer assembly)and an appropriate receptor for a target analyte on a support. Thesupport can be a microsphere or nanoparticle, a membrane, cuvette wallor the surface of a microtiter plate or glass slide, or any surface thatmay be interrogated by continuous or intermittent sampling(illumination/detection). The direct advantage of this approach is thatin each case, the superquenching occurs due to a specificligand-receptor interaction. Several different examples are discussed inthe following sections. Further, the assays may be carried out inoperationally different formats depending upon the specificrequirements.

Displacement Competition Assay

In the Displacement Competition Assay, the anchored fluorescentpolymer-receptor is pretreated with the QTL conjugate, resulting in thebinding of the QTL conjugate to the receptor and concurrentsuperquenching of the fluorescent polymer. As shown in FIG. 5, theactual analysis involves the addition of an analyte to the ensemble. Thefluorescence of the polymer increases quantitatively (turn on) with thelevel of the target agent in the analyte sample. Suitable examplesinclude proteins, viruses, bacteria, spores, cells, microorganisms,antibodies, antibody fragments, nucleic acids, and toxins. In thisexample, the assay may be homogeneous and the actual time for the assaymay be controlled by the “off rate” of the QTL from the receptor.

Direct Competition Assay

As shown in FIG. 6, in the Direct Competition Assay, the anchoredfluorescent polymer-receptor is treated with a mixture containing ananalyte (an unknown amount of the target agent) and a known amount ofQTL conjugate. The polymer fluorescence is quenched to an extentdetermined by the QTL:target agent concentration ratio. The stronger thefluorescence, the higher the concentration of the target agent. Anadvantage of this approach used is that the assay may be bothhomogeneous and near instantaneous. Since both the target agent and theQTL conjugate compete directly for “open” receptor sites, the responsecan be very rapid.

In another formulation, the anchored fluorescent polymer-receptor isincubated with an analyte sample before the fluorescence intensity ofthe sample is measured. The sample is then treated (following rinsesteps as necessary) with an excess of a QTL conjugate. The initialreading of fluorescence following treatment with the QTL conjugate showsquenching due to binding of the QTL conjugate to unoccupied receptorsites. The stronger the initial fluorescence quenching, the smaller thelevel of target agent. Monitoring the polymer fluorescence as a functionof time provides additional confirmation of the binding of the targetagent and its replacement by the QTL conjugate at the receptor.

A “Turn On” Competitive Assay Based on Polymer-Biomolecule Combinations.

Polymers that contain reactive end groups (e.g., polymer 10) may becovalently linked to a variety of materials, including small molecules,other polymers, and biomacromolecules. The resulting “hybrid molecule”may have similar solubility and will generally have the same ability asthe individual polyelectrolyte component to adsorb to a surface. Thesesurfaces include slides or plates, oppositely charged polymer beads(such as, but not limited to, quaternary amine-derivatized polystyreneor sulfonated polystyrene), natural or synthetic inorganic supports suchas clays or silica, charged membranes, semiconductor nanocrystals, andother porous materials. Thus, either independently or as a component ofa mixture, the use of a hybrid molecule can afford the preparation of asupported assembly containing a highly fluorescent species subject tosuperquenching. The hybrid molecule may also be employed in asolution-phase assay.

In one example, the carboxyl or amine terminus of an amino acid polymersuch as polymer 10 may be linked to a primary amine of a protein orantibody or antibody fragment to give a fluorescent compound 23. (SeeFIG. 7). This compound can either be used in solution or can bedeposited on a surface such as is described above. In either format, thebiomolecule portion of compound 23 should retain its specificrecognition function. Thus, treatment of compound 23 with a QTLbioconjugate results in formation of a complex that allows the quenchingcomponent to extinguish the fluorescence from compound 23. The exposureto molecules such as L or L′ that can compete with the QTL binding sitecan result in displacement of the bound QTL bioconjugate and a turningon of the fluorescence from compound. The most effective utilization ofcompound 23 will generally be on a surface or bead or other supportedformat where the aggregation of the fluorescent species can result inenhanced superquenching sensitivity. The hybrid molecule thus serves asa molecular or supramolecular (in supported formats) sensor whosefunction is shown schematically in FIG. 7.

In another example, a sensor/assay may be achieved in a supported formatby collecting individual (i.e., not covalently linked) polymer andbiomolecule components on the same bead, particle, or nanostructure. Forexample, carboxyl functionalized beads or particles may be used both tocovalently bind a protein, antibody, or antibody fragment via an aminegroup on the protein (as described above) and to bind a monomer (such as15), oligomer or polymeric fluorescent dye such as 10 by adsorption orcovalent attachment. Provided there is no significant quenchinginteraction between the dye ensemble and the biomolecule, the “dualcoated” beads will be strongly fluorescent. Here again, a competitivefluorescence “turn-on” assay may be constructed by the use of a QTLbioconjugate that associates with the biomolecule. Further, the additionof the QTL bioconjugate will result in a quenching of the dye ensemblefluorescence. Addition of a reagent L or L′ that can compete with theQTL bioconjugate for the binding site will result in the expulsion ofthe QTL molecule from the bead or particle and an increase (orunquenching) of the dye ensemble fluorescence. Because the spatial rangefor quenching is increased, a preferred embodiment will be the casewhere Q is an energy transfer quencher. This will allow the quenching ofall polymers within the Foerster transfer radius of the receptor-boundQTL molecule. For polymers bound on surfaces, this radius can beapproximately 100 Angstroms or more.

The dual coated beads or particles can also be used in a fluorescence“turn-off” competitive or noncompetitive assay. Treatment of the beads(initially uncomplexed) with an antigen (L or L′) will result in thebinding of the antigen to the biomolecule, but with negligiblefluorescence changes. Addition of an aliquot of a QTL molecule that canbind, but not compete with L or L′ will result in a quenching of thepolymer fluorescence in a “turn-off” response, that is proportional tothe number of receptor sites not occupied by the antigen. A QTL moleculethat can compete with antigen L or L′ will give a time-dependentresponse which can be used to measure both the level of antigen presentand the strength of its binding to the biomolecule.

The central component of the above-mentioned assays is the supported(and co-located) fluorescent polymer-receptor ensemble. They may beconstructed (but is not limited to) as outlined in the followingexamples. In the first example, a receptor, or “receptor binding site”is covalently attached to a support. Subsequently a fluorescent polymermay be adsorbed onto the same support as illustrated in FIG. 8. Examplesof receptors that may be covalently attached include proteins such asavidin, neutravidin or streptavidin or antibodies, peptides and nucleicacids. The degree of loading of both fluorescent polymer and receptorcan be controlled to obtain sensors having varied sensitivity anddynamic range. In a second example, as shown in FIG. 9, both the polymerand receptor may be covalently tethered to the support. In anotherformulation, illustrated in FIG. 10, a polymer or oligomer doped with areactive group is tethered to a receptor by a covalent linkage andadsorbed to a support. The polymer may be first adsorbed and thencovalently linked to the receptor or vice versa. To take advantage ofenhanced superquenching provided by “self-assembled” polymers, thefluorescent “polymer” ensemble can be constructed from monomers that maybe collected by either self-assembly (adsorption) or covalent linkage.Depending upon the requirements of the assay and the component “polymer”and receptor, the receptor may be covalently linked to the supportbefore or following generation of the self-assembled polymer.

In addition to the assays based on direct binding of a QTL conjugate tothe fluorescent polymer-receptor ensemble, assays may also beconstructed based on secondary recognition events. For example, thecurrent platforms can be extended to a sandwich format in which a targetagent having multiple binding sites for the same or other receptor issensed. This format is illustrated in FIG. 11. Binding of the targetagent to a receptor site causes little or no change in the fluorescentpolymer fluorescence. However, addition of a QTL conjugate which alsobinds to the receptor results in bringing the quencher close enough toquench the fluorescence in a direct assay. Such a sandwich assay can beadapted to sense a variety of agents including bacterial spores.

Having generally described the invention, a further understanding can beobtained by reference to certain specific examples provided herein forpurposes of illustration only and are not intended to be limiting unlessotherwise specified.

EXAMPLES Example 1

Commercial polystyrene beads containing streptavidin covalently tetheredto the surface (0.53 micron microspheres purchased from BangsLaboratories, Inc., Fishers, Ind.) were coated with the anionicconjugated polymer 24, a derivative of poly(phenyleneethynylene) (PPE)(structures 24-27 are shown in FIG. 12). The level of loading of 24 onthe surface can be controlled depending on the loading of the polymer.The number of biotin binding sites (maximum biotin-FITC bindingcapacity=1.42 ug/mg of microspheres) is also variable and controllable.For an initial assessment of the ability of the coated microspheres tofunction in biosensing, a QTL conjugate formed of an energy transferquencher (Alexafluor 594, purchased from Molecular Probes) wasconjugated to the streptavidin ligand biotin. In separate studies it wasdemonstrated that nonspecific quenching of the polymer fluorescence bynon-biotinylated Alexafluor 594 does not occur. Depending on the levelof coating, the K_(SV) was found to vary between 3×10⁷ and 3×10⁸ M⁻¹over two logs of QTL concentration. The level of the QTL detected bydirect binding to the receptor in a conventional 96-well plate was lessthan 100 femtomoles. For this assay, it was determined that anintermediate level of polymer loading onto the beads gave optimalinitial quench sensitivity and a wide dynamic range. (See FIG. 13).

To generalize the assay using these beads, biotinylated antibodies canbe used to tether specific receptors. The binding of the biotinylatedantibodies produces little change in the fluorescence of the polymer.However, the addition of a conjugate recognized by the antibody andcontaining an energy transfer quencher does result in quenching of thepolymer fluorescence. Thus, as shown in FIG. 14, it has beendemonstrated that a biotinylated capture antibody can bind to anantibody-based QTL conjugate (target antibody derivatized with an energytransfer quencher) and be detected at levels less than one picomole).

From this example, it is evident that the same beads can be used toconstruct a wide array of assays based on antibody-antigen interactions.In the general case, two additional components are required: abiotinylated antibody or other receptor and a QTL conjugate that isrecognized by the antibody. All three of the assay paths described abovecan be used with these beads. The use of labeled beads (e.g., apolystyrene bead labeled in the interior of the bead with a fluorescentdye tag having distinct fluorescent wavelengths) or different polymerswith different antibodies or receptors allows for the simultaneous assayof multiple target analytes.

Example 2

A somewhat lower molecular weight PPE oligomer, monofunctionalized withcarboxylate 25, was adsorptively coated on quaternaryammonium-derivatized polystyrene microspheres. Following deposition,rabbit anti-goat IgG antibodies were covalently linked to the polymerthrough the available carboxyl functionality. The fluorescence of thepolymer remained strong following the antibody coupling and showedlittle sensitivity toward photobleaching. However, the fluorescence ofthe ensemble of oligomers was quenched specifically by the addition ofgoat anti-rabbit IgG conjugated to the fluorescent energy transferquencher, Alexafluor 532. Fluorescence quenching could be detected at<500 fmole levels in a 96-well plate format. (See FIG. 15).Additionally, goat anti-rabbit IgG antibodies coupled with thenonfluorescent energy transfer quencher QSY35 also exhibited quenchingon association with the bead-anchored polymer-antibody receptor. In thiscase, a K_(SV)=8×10⁷ M⁻¹ was measured in the sub to few picomolesconcentration range.

Example 3

Cyanine dyes exhibit induced J-aggregation on anionic nanoparticles andmicrospheres. For simple cyanine monomers such as 26, adsorption ontoclay or silica particles is reversible and thus individually coatedparticles coated with different cyanine dyes or cyanine mixtures exhibitexchange among the cyanines. It has been determined that the use ofamphiphilic cyanine dyes such as the derivative of 26 where the N-ethylgroups have been replaced by N-octadecyl groups results in moleculesthat can be irreversibly adsorbed onto silica microspheres. Thus,individual amphiphilic cyanine dyes or mixtures of amphiphilic cyaninesmay be coated separately onto silica microspheres and then mixed withsilica microspheres coated with other formulations of cyanineamphiphiles. The mixtures show no evidence of exchange of cyaninesbetween different particles and thus permit the use of these mixturesfor the simultaneous sensing of multiple agents. The use of energyaccepting amphiphilic guests such as the corresponding amphiphiliccyanine to 4 results in the same emission wavelength shifting andaffords the construction of several ensembles capable of emittingfluorescence at different wavelengths from the same host amphiphiliccyanine.

The fluorescence of the aggregated cyanine dye may be quenched by eithercationic or anionic energy accepting cyanine dyes or by electrontransfer quenchers. This quenching can be tuned by varying the level ofcoating of the cationic cyanine dye on the anionic nanoparticle ormicrosphere. At low loading of the particle with a cationic cyanine, theparticle has regions of exposed negative charge and positively chargedquenchers are attracted (and exhibit high superquenching constants)while potential anionic quenchers show low quenching via thesenonspecific interactions. At high loading of the particles, thesituation is reversed and anionic quenchers show attractive butnonspecific interactions and consequent high quenching constants whilecationic quenchers are ineffective. For clay nanoparticles, optimumresults occur with near 100% coverage of the clay surface by a cyanineor cyanine mixture. At this level of coverage, selective quenching byanionic quenchers occurs. For cyanine dye aggregates on the claynanoparticles, the most effective quenching occurs when like-chargedcyanines are co-adsorbed.

For example, the addition of energy accepting cationic cyanine 27 toexcess cyanine 26 results in 50% quenching when the ratio of compound 26to compound 27 ratio is 400:1. The quenching of 26 by 27 results in thesensitized emission of 27 and offers a potential advantage in separatingthe excitation and emission of the nanoparticle-supported ensemble.These particle-bound “self-assembled polymers” offer a convenientplatform for sensing similar to those discussed above in Example 1 and2. Coating of cyanine monomer or a mixture (such as 26 and 27) ontoanionic microspheres or nanoparticles that already have a covalentlyanchored receptor such as streptavidin or an antibody can result in theformation of regions of J-aggregate or mixed aggregate on all accessibleanionic surfaces of the support. This renders the ensemble overallslightly cationic and therefore of very low susceptibility tononspecific association with cationic quenchers. However, cationic QTLconjugates can associate with the particles by specific ligand-receptorinteractions in the same ways as described in the Examples 1 and 2above. Thus, the superquenching of the self-assembled polymers can beharnessed in improved biosensing through specific association in theco-located receptor-self-assembled polymer ensembles.

Example 4

The same kind of self-assembled polymers may also be constructed bycovalent linkage of cyanine (or other monomers) onto a denselyfunctionalized surface. As shown in FIG. 16 a, the same cyaninechromophore present in 26 can be constructed by covalent attachment intwo stages. It has been determined that amine functionalized silicamicrospheres can form a platform onto which a high level of coverage canbe obtained. For microspheres coated only with the monomer, it is foundthat, depending on the surface derivatization and reaction conditions,different populations of at least three species are obtained. The firstspecies has absorption and fluorescence close to those of the monomer. Asecond, longer-wavelength absorbing species shows very similarabsorption and emission to the J-aggregate of 26 described above. Thethird species exhibiting a somewhat broadened emission at longerwavelengths is usually not prominent in absorption but frequentlyincludes the predominant emission, regardless of the wavelength at whichthe mixture is excited. It has been found that quenching by non-specificinteractions can be observed for anionic electron transfer dyes(AQS-Biotin (5) (FIG. 2), K_(SV)=3×10⁷ M⁻¹) and for a cationic energytransfer dye (QSY-21 (6) (FIG. 17), K_(SV)=5.3×10⁸ M⁻¹) at subpicomolelevels of quencher. In order to construct a sensor analogous to thosedescribed in the Examples above, the covalently-linked cyanine wasconstructed with varying amounts of an additional functionalized sitecontaining a carboxyl group as shown in FIG. 16 b. Once the dye has beentethered to the surface, the carboxyl sites may be used to append areceptor as outlined in Example 2 set forth above. The appending of areceptor on the surface of the covalently tethered “self-assembledpolymer” has the advantage of shielding the dye from non-specificassociation with potential quenchers and restricting quenchinginteractions to QTL conjugates associating specifically with thereceptor.

The invention of this application has been described above bothgenerically and with regard to specific embodiments. Although theinvention has been set forth in what is believed to be the preferredembodiments, a wide variety of alternatives known to those of skill inthe art can be selected within the generic disclosure. The invention isnot otherwise limited, except for the recitation of the claims set forthbelow.

REFERENCES

1. L. Chen, D. W. McBranch, H.-L. Wang, R. Helgeson, F. Wudl and D. G.Whitten, “Highly-Sensitive Biological and Chemical Sensors Based onReversible Fluorescence Quenching in a Conjugated Polymer”, Proc. Nat'lAcad. Sci. USA, 96:12287 (1999).

2. L. Chen, D. McBranch, R. Wang and D. G. Whitten, “Surfactant-InducedModification of Quenching of Conjugated Polymer Fluorescence by electronAcceptors: Applications for chemical Sensing”, Chem. Phys. Lett.,330:27-33 (2000).

3. L. Chen, S. Xu, D. McBranch and D. G. Whitten, “Tuning the Propertiesof Conjugated Polyelectrolytes Through Surfactant Complexation”, J. Am.Chem. Soc., 122:9302-9303 (2000).

4. D. Whitten, L. Chen, R. Jones, T. Bergstedt, P. Heeger, D. McBranch,“From Superquenching to Biodetection; Building Sensors Based onFluorescent Polyelectrolytes” in “Molecular and SupramolecularPhotochemistry, Volume 7: Optical Sensors and Switches”, Marcel Dekker,new York, eds. V. Ramamurthy and K. S. Schanze, Chapter 4, pp 189-208(2001).

5. R. M. Jones, T. S. Bergstedt, C. T. Buscher, D. McBranch, D. Whitten,“Superquenching and its applications in J-aggregated cyanine polymers”,Langmuir, 17:2568-2571 (2001).

6. L. Lu, R. Helgeson, R. M. Jones, D. McBranch, D. Whitten,“Superquenching in cyanine pendant poly-L-lysine dyes: dependence onmolecular weight, solvent and aggregation”, J. Am. Chem. Soc., in press.

7. R. M. Jones, T. S. Bergstedt, D. W. McBranch, D. G. Whitten, “Tuningof Superquenching in layered and mixed fluorescent polyelectrolytes”, J.Am. Chem. Soc., 123:6726-6727 (2001).

8. R. M. Jones, L. Lu, R. Helgeson, T. S. Bergstedt, D. W. McBranch, D.Whitten, “Building highly sensitive dye assemblies for biosensing frommolecular building blocks”, Proceedings Nat'l. Acad. Sci. USA,98:14769-14772 (2001).

1. A composition of matter comprising: a fluorescent polymer affixed toa support; and a receptor specific for a target biological agent affixedto said support, said receptor being adapted for complexation with achemical moiety comprising (a) a recognition element capable of bindingto said receptor, (b) a tethering element, and (c) a property-alteringelement such that when said chemical moiety is bound to said receptor,said property-altering element is located sufficiently close to saidfluorescent polymer such that the fluorescence emitted by saidfluorescent polymer is altered from that emitted when said bindingbetween said receptor and chemical moiety does not occur.
 2. Thecomposition of matter of claim 1, wherein said polymer is anchored tosaid support via adsorption or a covalent linkage.
 3. The composition ofmatter of claim 1, wherein said polymer is selected from the groupconsisting of a neutral polymer and a charged polymer.
 4. Thecomposition of matter of claim 1, wherein said polymer compriseschromophores which are covalently or non-covalently linked and aggregateto form a polymer ensemble.
 5. The composition of matter of claim 4,wherein said chromophores are similar.
 6. The composition of matter ofclaim 4, wherein said chromophores are dissimilar.
 7. The composition ofmatter of claim 4, wherein said polymer ensemble is covalently linked toa second polymer.
 8. The composition of matter of claim 1, wherein saidpolymer and receptor are covalently bound to said support.
 9. Thecomposition of matter of claim 1, wherein said receptor is covalentlybound to said polymer.
 10. The composition of matter of claim 1, whereinsaid fluorescent polymer is a conjugated or J-aggregated polymerassembly comprising assembled monomers or oligomers.
 11. The compositionof matter of claim 1, wherein said polymer is selected from the groupconsisting of conjugated polyelectrolytes, functionalized conjugatedoligomers, uncharged conjugated polymers, charged conjugated polymersand conjugated polymer blends.
 12. The composition of matter of claim 1,wherein said recognition element is selected from the group consistingof chemical ligands, hormones, antibodies, antibody fragments,oligonucleotides, antigens, polypeptides, glycolipids, proteins, proteinfragments, enzymes, peptide nucleic acids and polysaccharides.
 13. Thecomposition of matter of claim 1, wherein said tethering element isselected from the group consisting of a single bond, a single divalentatom, a divalent chemical moiety, a multivalent chemical moiety,polyethylene, polyethylene oxides, polyamides and non-polymeric organicstructures.
 14. The composition of matter of claim 1, wherein saidproperty-altering element is selected from the group consisting ofmethyl viologen, quinones, metal complexes, fluorescent dyes,non-fluorescent dyes, electron accepting moieties, electron donatingmoieties and energy accepting moieties.
 15. The composition of matter ofclaim 1, wherein said support is selected from the group consisting ofstreptavidin coated spheres, polymer microspheres, silica microspheres,organic nanoparticles, inorganic nanoparticles, magnetic beads, magneticparticles, semiconductor nanoparticles quantum dots, membranes, slides,plates and test tubes.
 16. The composition of matter of claim 1, whereinsaid target biological agent is selected from the group consisting ofproteins, viruses, bacteria, spores, cells, microorganisms, antibodies,antibody fragments, nucleic acids and toxins.
 17. An assay for detectingthe presence of a target biological agent in a sample comprising:determining the fluorescence emitted by said chemical composition ofclaim 1 in the absence of a sample, contacting said sample and saidchemical moiety with said receptor; and determining the fluorescenceemitted by said fluorescent polymer after said contacting step; whereina difference in fluorescence emitted after said contacting step comparedto that emitted in the absence of said sample is indicative of thepresence of said biological agent.
 18. The assay of claim 17, whereinthe amount of target biological agent present in said sample iscorrelated with the amount of said difference in fluorescence.
 19. Theassay of claim 17, wherein said chemical moiety is added to saidchemical composition prior to said sample.
 20. The assay of claim 17,wherein said chemical moiety and said sample are simultaneously added tosaid chemical composition.
 21. The assay of claim 17, wherein saidsample is added to said chemical composition prior to said chemicalmoiety.
 22. The assay of claim 17, wherein said sample and chemicalcomposition are incubated prior to the addition of said chemical moiety.23. The assay of claim 17, wherein said target biological agent isselected from the group consisting of proteins, viruses, bacteria,spores, cells, microorganisms, antibodies, antibody fragments, nucleicacids and toxins.
 24. A composition of matter comprising: a fluorescentpolymer affixed to a support; and a chemical moiety affixed to saidsupport, said chemical moiety comprising a receptor specific for atarget biological agent and a QTL bioconjugate including (a) arecognition element which binds to said receptor, (b) a tether, and (c)a property-altering element located sufficiently close to saidfluorescent polymer such that the fluorescence emitted by saidfluorescent polymer is altered when said property-altering element andsaid fluorescent polymer are complexed together to a distinguishabledegree, said QTL bioconjugate being susceptible of subsequent separationfrom said polymer upon exposure to an agent having an affinity forbinding to said receptor, wherein the separation of said QTLbioconjugate from said receptor results in a detectable change influorescence of said polymer.
 25. An assay for detecting the presence ofa target biological agent in a sample comprising: (a) determining thefluorescence emitted by said chemical composition of claim 1 in theabsence of a sample; (b) contacting said chemical composition of step(a) with said chemical moiety; (c) determining the fluorescence emittedby said fluorescent polymer after said contacting step (b); (d)contacting said chemical composition of step (a) with said chemicalmoiety and said sample; and (e) determining the fluorescence emitted bythe fluorescent polymer after said contacting step (d); wherein adifference in fluorescence after said contacting step (d) compared tothat emitted after said contacting step (b) is indicative of thepresence of said target biological agent.
 26. The assay of claim 25,wherein the amount of target biological agent present in said sample iscorrelated with the amount of said difference in fluorescence.
 27. Theassay of claim 25, wherein in step (d), said chemical moiety is added tosaid chemical composition prior to said sample.
 28. The assay of claim25, wherein in step (d), said chemical moiety and said sample aresimultaneously added to said chemical composition.
 29. The assay ofclaim 25, wherein in step (d), said sample is added to said chemicalcomposition prior to said chemical moiety.
 30. The assay of claim 25,wherein in step (d), said sample and chemical composition are incubatedprior to the addition of said chemical moiety.
 31. The assay of claim25, wherein said target biological agent is selected from the groupconsisting of proteins, viruses, bacteria, spores, cells,microorganisms, antibodies, antibody fragments, nucleic acids andtoxins.